Allergen desensitization method

ABSTRACT

The present application relates to a method for desensitization of allergic patients. More specifically it relates to an epicutaneous desensitization method, applicable to any type of allergens and of patients. The method of the invention is essentially non-invasive and does not require the use of adjuvants. Further, it may be easily applied and monitored by the actual patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/745,870, filed Jun. 2, 2010, which is a U.S. national stage filingunder 35 U.S.C. §371 of International Patent Application No.PCT/FR2008/052199, filed Dec. 3, 2008, which claims the benefit of U.S.Provisional Patent Application No. 61/084,305, filed Jul. 29, 2008, andFrench Patent Application No. 0759503, filed Dec. 3, 2007, the entirecontents of each of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present application relates to a method for desensitizing allergicpatients. More specifically it relates to an epicutaneousdesensitization method, applicable to any type of allergens andpatients. The method of the invention is essentially non-invasive anddoes not require the use of adjuvants. Further, it may easily be appliedand followed by the actual patient.

DESCRIPTION OF RELATED ART

Allergy occupies an increasingly large place in daily medical practice.It is a worldwide public health phenomenon classified as the fourthworldwide scourge by the WHO. In France, one Frenchman out of three isallergic and both respiratory and food allergy affects an increasingportion of the population of adults and children.

Desensitization is the technique according to which by administeringminimal amounts of allergens it is possible to more or less suppress theallergic phenomenon. Since the beginning of the years 2000, it has beenrecognized by the WHO as the only method for basically treating allergy.

It is now established that desensitization affects the immune responsetowards allergens as soon its first steps (Tij et al., 2004), whichmeans that it not only reduces the symptoms related to short termallergic reaction but also that it modifies the natural history of theallergic <<disease>> and that it prevents both occurrence of allergy tonew allergens, but also progression of the symptomatology towards moresevere clinical signs, such as for example the transformation ofallergic rhinitis into asthma.

Immunotherapy has proved to be effective in patients affected withsevere IgE-dependent allergy, sensitized to a restricted number ofallergens.

Although the action mechanisms of treatments by immunotherapy are notyet well-known, the latter may act by:

-   -   increase in the IgGs and in particular in IgG4 fractions,        antibodies blocking in vitro the biological effects of the IgEs,        even if the significance of these effects in vivo still has to        be evaluated,    -   alteration of the TH1/TH2 balance, promoting TH1 response,    -   production of T cells producing interleukin 10 (IL-10). The        latter has many anti-allergic properties against mastocytes,        certain T lymphocytes and eosinophilics and also promotes the        production of IgG4.

Immunotherapy via a subcutaneous route represents for mostallergologists the standard route and it is still widely used. Althoughcostly and requiring the intervention of a specialist physician at eachinjection, it is today still considered as the standard desensitizationroute in children (Pajno et al., 2005). However it is not without risks,since the frequency of deaths is estimated to be 1 for 2.5 millioninjections with an average of 3.4 deaths per year.

Certain anaphylactic allergens with a high risk of reaction are not usedwithin the scope of subcutaneous desensitization cures. This is the caseof groundnuts and of most food allergens.

Sublingual immunotherapy is now considered by the WHO as a satisfactoryalternative to immunotherapy via a subcutaneous route. It is beingevaluated in many countries.

The first commercial preparations of tablets to be administered underthe tongue, intended for a wide public are being sold in certainEuropean countries and are being pre-marketed in the United States.

Much better tolerated by the patients, sublingual immunotherapy is formany authors, of a better cost-effectiveness ratio than the other routesof administration since it does not require the intervention of aphysician and may be self-administered by the patients which admit thatthey prefer it to the other routes (Pajno et al., 2005).

Adverse reactions are rare during treatments via a sublingual route,estimated to be from 0.1 to 0.2 reactions for 1,000 administered doses.The reactions are mainly minor, affecting the buccal cavity or thesublingual area or the gastro-intestinal sphere (La Rosa et al., 1999).

In terms of effectiveness, the sublingual route is however considered bycertain persons as less effective than the subcutaneous route; itrequires the use of a larger amount of allergen and cannot be authorizedwith all allergens even if tests via this route for desensitization togroundnuts and to cow milk are in progress. The safety of the use ofthis technique with food allergens remains to be evaluated.

Intra-nasal immunotherapy has proved to be effective in 17 controlledstudies out of 18. This is therefore an effective and safe route at thevery least during allergic rhinitis. However, it is generally poorlytolerated by patients, which increasingly limits its clinical use. In astudy comparing different desensitization routes, treatments via a nasalroute were prematurely interrupted in close to 50% of the cases beforeone year, while early stopping only concerned less than 10% of thechildren treated via a subcutaneous or sublingual route (Pajno et al.,2005).

An advantageous alternative to these desensitization methods would liein the possibility of carrying out immunotherapy via the epicutaneousroute, i.e. by repeated application of an allergen on the skin,typically leading to diffusion of the allergen in the surface layers ofthe skin, generally without any significant transcutaneous passage.

It has been demonstrated for a long time in animals that repeatedapplication of an allergen on the skin is capable of causingsensitization of the animal to said allergen (Golovanoff, 1926). Theobtained reaction is of the systemic type and affects the body in itswhole and therefore well beyond the simple cutaneous sphere.

More recently, models of mice sensitized to cow milk via an epicutaneousroute have been developed (Chang). It has been shown that in thesecases, the elicited immune response involves different cell populationsand results in the activation of TH2 lymphocytes, which promote theproduction of specific IgE type antibodies of the allergen.

Certain investigations were able to establish that if the spontaneousresponse of the skin under stimulation conditions was actually of theTH2 type, it was possible to modify the immune profile of the responseby subjecting the skin to different stimuli:

-   -   Aggressions by physical agents such as ultraviolet rays    -   Mechanical aggression of the skin by repeated strippings        modifying the structure of the corneal layer    -   Aggression by microbial agents such as choleric toxin    -   Modification of skin permeability.

The type of immune response to the contact of an allergen may bemodified by these different agents and results in preferentialactivation of TH1 lymphocytes promoting the production of antibodies ofthe IgG4 type.

The epicutaneous route was used by allergologists during the 50ies. Itconsisted of carrying out scarification on the skin through a drop ofallergen several times a week (Pautrizel et al., 1957). Carried out byan experienced allergologist, this technique provided effectiveness andsafety. However, carried out on a very small scale, and not verystandardized, it has remained not very widespread. Finally, the hardshipgenerated by repeated scarifications often very extended and mutilatingis undoubtedly the reason why this method is abandoned today.

Patent application EP 1031346 proposes a vaccine containing atransdermal device and an antigen or allergen in order to obtaindesensitization and application WO 2007/122226 mentions the use of acutaneous device for desensitizing an allergic subject.

SUMMARY OF THE INVENTION

The present invention lies in the development of an effectiveepicutaneous desensitization method and with which the drawbacks andlimits of the prior technique may be overcome.

More specifically, the invention partly lies on the demonstration thatthe specific inflammatory response caused by cutaneous application ofallergens largely influences and participates in the reaction of immunecells in the sense of tolerance.

The invention consists of applying an allergen in a repeated andprolonged way on the skin so as to cause an inflammatory reaction, theprofile of which oriented by the duration and the repetition of theapplications is of the <<tolerogenic>> type. Thus, the invention is notonly directed to eliciting the inflammatory reaction but also toorienting it.

The present application shows that controlling this inflammatoryreaction conditions the obtained degree of tolerance and represents anessential element of the epicutaneous desensitization process. Theinvention shows that repeated elicitation of this specific inflammatoryreaction is important for gradually inverting the immune response in thesense of tolerance, and it makes epicutaneous desensitization possible,even in the absence of any adjuvant or pre-treatment of the skin.

The invention thus proposes for the first time, a cutaneousdesensitization method comprising repeated cutaneous application of anallergen on the skin of a subject under conditions allowing theinflammation generated by the application of the allergen on the skin tobe sustained during the treatment. Advantageously, the method comprisesat least one step for monitoring and/or evaluating the degree of theinflammation generated by the application of the allergen on the skin,the elicitation, sustainment and/or control of this reaction giving thepossibility of directing the reaction of the body to the allergen in thesense of tolerance.

An object of the invention thus lies in the use of an allergen forpreparing a composition for epicutaneous desensitization of a subjectallergic to said allergen, by repeated applications of the allergen onthe skin of a subject, characterized in that the allergen is applied onthe skin under conditions allowing an inflammatory reaction to besustained during the treatment. The invention indeed shows thatsustainment of this inflammatory reaction promotes a response of thetolerogenic type from the immune system towards the allergen.

Another object of the invention lies in the use of an allergen forpreparing a composition for epicutaneous desensitization of a subjectallergic to said allergen, by repeated applications of the allergen onthe skin of a subject, characterized in that it comprises during thetreatment, preferably during each application of the allergen, a stepfor controlling and/or sustaining the inflammatory reaction generated bythe application of the allergen on the skin.

Another object of the invention lies in the use of an allergen forpreparing a composition for epicutaneous desensitization of a subjectallergic to said allergen, by repeated applications of the allergen onthe skin of a subject, characterized in that it comprises during thetreatment, control and sustainment of the inflammatory reactiongenerated by the application of the allergen on the skin.

Another object of the invention lies in the use of an allergen forpreparing a composition for modifying in the sense of tolerization theimmune response of a subject allergic to said allergen, comprisingrepeated application of the allergen on the skin of a subject underconditions allowing sustainment of an inflammatory reaction during thetreatment.

The invention further relates to an epicutaneous desensitization methodof a subject allergic to an allergen, by repeated application of theallergen on the skin of a subject, characterized in that the allergen isapplied on the skin under conditions allowing sustainment of aninflammatory reaction during the treatment.

As this will be described in details subsequently in the application,the inflammatory reaction is advantageously sustained by controlling ormodulating during the treatment the dose or the frequency or theapplication time of the allergen, and/or its nature.

Another object of the invention lies in an epicutaneous desensitizationmethod of a subject allergic to an allergen, characterized in that itcomprises at least one step for evaluating during the treatment thedegree of the inflammation generated by the application of the allergenon the skin. By evaluating the degree of the inflammatory reaction, theconditions of desensitization may be adjusted if necessary.

The invention further relates to a method for desensitizing a subjectallergic to an allergen, by repeated applications of the allergen on theskin of a subject, characterized in that it comprises, at eachapplication of the allergen, a step for controlling the inflammatoryreaction.

The invention further relates to a method for desensitizing a subjectallergic to an allergen, by repeated application of the allergen on theskin of a subject, characterized in that it comprises sustainment,during the treatment, of the inflammatory reaction generated by theapplication of the allergen on the skin.

The invention also relates to the use of the inflammatory reactiongenerated by the application of an allergen on the skin of a patient asa marker in order to adapt the treatment of said patient forepicutaneous desensitization to said allergen.

As this will be described subsequently in the application, the inventionis applicable to any type of allergen and may be applied with anycutaneous application device. It allows effective desensitizationwithout it being necessary to treat the skin of the patient beforehand.Moreover, it does not require co-administration of adjuvant compounds.

The use of allergen deposited on the skin of an allergic subject isperfectly safe, as demonstrated by the cumulated experience in thediagnostic field with the atopy patch test. In our experience in Francewith Diallertest®, a first ready-to-use atopy patch test marketed since2004, no adverse effect has been reported, whether this is a severelocal action or a generalized reaction of the systemic type.

After application of an allergen on the skin, the immune system mayeither not react or react with local (cutaneous) inflammatory reactionof a systemic immune reaction.

In a particular embodiment of the invention, the application of anallergen on the skin of a subject allergic to said allergen induces acutaneous reaction, in particular of the inflammatory type, triggered bythe contact of the allergen with keratinocytes and Langerhans cellspresent at the surface of the skin.

During epicutaneous immunotherapy by the method of the invention, thisreaction caused by the application of the allergen on the skin of asubject allergic to said allergen is sustained and/or controlled duringthe cure. The invention indeed shows that sustainment and control ofthis reaction result in gradual orientation of the immune response inthe sense of tolerance of the subject to said allergen. This response isclinically materialized by a gradual reduction or complete extinction ofthe cutaneous reactivity and by an increase in the tolerance thresholdto said allergen.

The mechanism stated above applies to any allergen, notably to allergensacting on the digestive sphere (food allergy, such as to ovalbumin,groundnuts, shellfish, etc.), on the pulmonary sphere (respiratoryallergy such as for example to pollen) or the cutaneous sphere.

Thus, the principle on which the method of the invention is based andwhich is perfectly illustrated by the experimental data discussedhereafter, is that it is possible to induce via a cutaneous route,tolerance of a subject previously sensitized to an allergen. This effectis obtained by maintaining the allergen on the skin of the subjectallergic to said allergen and by doing this, causing, sustaining andcontrolling the inflammatory reaction to the allergen with the purposeof directing the reaction of the body to the allergen in the sense oftolerance.

Thus, the epicutaneous immunotherapy method of the invention is atherapeutic method for allergy which consists of putting immune cells ofthe skin (e.g. Langerhans cells, mastocytes, macrophages, leukocytes) incontact with an allergen in a subject allergic to said allergen and ofeliciting via the specific, controlled and sustained cell inflammatoryreaction, a conversion of the immune response resulting in tolerance tosaid allergen by the allergic patient. In the method of the invention,and unlike all the prior approaches to desensitization, thedesensitization process is monitored and guided by the nature and thedegree of the inflammatory reaction of the patient This approachprovides effective desensitization adjustable to the needs and to thecharacteristics of each patient, and does not require the use ofadjuvant compounds or preliminary perforating or abrasive treatment ofthe skin.

DETAILED DESCRIPTION

The method of the invention generally comprises application in arepeated, prolonged and controlled way of an allergen on the skin of asubject allergic to said allergen, under conditions (for example doseand/or frequency and/or application time conditions) allowingsustainment of an inflammatory reaction specific to the allergen duringthe treatment. In an advantageous embodiment, the application isaccomplished without any adjuvant compound and/or on a healthy skin.

The method of the invention generally comprises the following phases:

-   -   a. optional selection of the patients and/or the allergen (dose,        nature, formulation, etc.);    -   b. application of the allergen to the patient; and    -   c. control of the inflammatory reaction; steps b. and c. being        advantageously repeated one or several times during the        treatment, the condition of the allergen (dose, nature,        formulation, application frequency, exposure time, etc.) being        adapted during the treatment in order to sustain the        inflammatory reaction.

Insofar that the invention is based on the control of the inflammatoryreaction, it is important that allergic patients subject to thetreatment have such a response during the first application of theallergen on the skin. For this, the patient is advantageously subject toa first application (series) of the allergen, so as to determine theallergen preparation giving the most suitable reactivity at thebeginning of the treatment.

For this purpose, the patient is advantageously subject to a firstapplication of the allergen, in a predefined form and single dose.

If the subject develops a suitable inflammatory reaction, the treatmentmay be engaged on the basis of this allergen dose/form. A suitablereaction for example refers to an inflammation reaction visible to thenaked eye, typically as infiltrated erythematous lesions either providedor not with papules or assuming an eczematiform aspect.

If the ascertained reaction is of strong intensity, including vesiclesor even phlyctena, or is widely disseminated in neighboring skin areasor is strongly pruriginous, the allergen concentration for initiatingthe cure will be reduced, in order to obtain a suitable response oralternatively the application time of the allergen on the skin will bereduced.

On the contrary, when the ascertained reaction is of too low intensity,the allergen doses and/or the application time and/or the number ofdevices applied simultaneously, will be increased in order to obtain aninitial significant reaction as defined above. In this case, it is alsopossible to test other forms of the allergen, in order to identifypreparations giving better reactivity of the subject. Thus, indeed, theallergen may exist under different forms (complete foodstuff, proteinextract, peptides, recombinant or synthetic products, etc.) which mayelicit a different inflammatory response in allergic patients. Thisfirst phase of the treatment may thus allow determination of the mostsuitable form of the allergen for the beginning of the cure.

In this respect, in a particular embodiment, the patient is subject atthe initial time of the cure to a series of devices containing variousconcentrations and/or forms of the allergen. With this first treatmentit is possible to determine the dose and/or the optimum form, on apatient basis, being used as a reference for initiating the cure. Ifnecessary, the use of these devices with various concentrations and/orforms may be repeated during the cure so as to adapt the dose/form usedduring treatment, depending on the change in reactivity of the subject.

When the initial dose/form has been determined, the epicutaneousimmunotherapy treatment may be engaged. This treatment comprisesrepeated application of the allergen on the skin. The application isadvantageously carried out at the upper portion of the back of thesubject or on the inner face of the arms or forearms. It may also becarried out on any other region of the body, under the preferentialcondition that the local reactivity to the allergen has been checkedbeforehand. It is understood that, during the treatment, the allergenmay be applied on different regions of the body. The applications arerenewed at a variable frequency, depending on the therapeutic procedureused (a typical procedure comprises an initial application timecomprised between about 6 and 72 hrs). They may be applied continuouslyor batchwise while observing variable time intervals between theapplications (which may range from 1 day to three weeks, for example).Repeated and prolonged application for several months of the allergenresults in local release of mediators of the inflammation and in theinflow of inflammatory cells which influence the reaction of the immunecells in the sense of tolerance. A typical procedure comprises repeatedapplication of the allergen, for periods from about 6 to 72 hrs, spacedout by about 5-15 d, over a period required for desensitizing thesubject, this period may exceed several months.

In order to ensure suitable desensitization, the present inventiondemonstrates that it is important to control and sustain (by modulatingthe doses and/or the times or frequency of exposure to the allergen, orits form), a local inflammation reaction during the treatment. Themethod of the invention thus comprises regular evaluation of the degreeof the inflammation generated by repeated application of the allergen onthe skin. Thus, during the cure, the allergen doses and/or the period,and/or the number of devices used simultaneously, will be adapteddepending on the degree of the cutaneous reactivity. A decrease in theintensity of the cutaneous reaction during the treatment results in anincrease in the application frequency and/or in the number of devicesapplied simultaneously and/or in the dose of the allergen, and/or in amodification of its form. On the contrary, an increase in the intensityof the cutaneous reaction results in a reduction of the applicationfrequency and/or of the number of devices applied simultaneously and/orof the concentration of the allergen in said device, and/or of its form.

The evaluation of the intensity of the inflammatory reaction, inparticular of the cutaneous reaction, may be carried out by thephysician and/or by the actual patient. This evaluation may be carriedout by any method, conveniently by visual examination or by contact, forexample by using the consensual assessment grid of the EAACI (seeAllergy 61 (2006) 1377-1384). Thus, there exist different degrees in thecutaneous inflammatory reaction: erythema-erythema andinfiltrations-erythema and a few papules-erythema provided with manypapules-erythema with vesicles. A suitable inflammatory reaction istypically an inflammatory reaction of type III, i.e. comprising erythemaand a few papules. As far as possible, this is the type of reactionwhich one attempts to sustain during the treatment. Of course, when thepatient develops a less strong reaction from the start, the referencereaction will necessarily be less strong, and it will be adapted on apatient basis. Moreover, during the treatment, the inflammatory reactionwill decrease because of the desensitization of the subject.

For applying the invention, it is also possible to evaluate theinflammatory reaction by dosing inflammation markers, such as cytokinesor interferons.

In order to adjust the treatment as best as possible, in a particularembodiment of the invention, the inflammatory reactivity is evaluatedone or several times during the treatment by putting the subject intocontact with a range of doses of the allergen, allowing selection of thebest adapted dose for continuation of the treatment.

The evaluation of the inflammatory reaction may be performed all alongthe treatment, i.e. at each new application of the allergen.

It is also possible to only perform this evaluation at certain intervalsand not at each application.

Further, if it is preferred to sustain an inflammatory reaction allalong the treatment, this is not mandatory and effective desensitizationmay also be obtained when the immune contact has been established afirst time and when specific cascade reactions (activation ofimmunologically competent cells and production of cytokines andantibodies) have been sufficiently triggered. Thus, in practice, whenthe inflammatory reaction has reached a sufficient level, for exampleexpressed by a cutaneous reaction of the intended intensity, it ispossible to remove the device, and then after a delay to be defined, toput another one in another cutaneous area.

In an alternative of the invention, the allergen (or the applicationdevice) is maintained on the skin at least until the inflammatoryreaction reaches a degree of reference. When this degree is reached, theallergen (or the device) may be removed, and a latency period isobserved before a new application. The reference reaction is typically areaction visible to the naked eye, for example as infiltratederythematous lesions or having an eczematiform aspect.

The allergen (or the device) may also be maintained on the skin so as tosustain the inflammatory reaction. Typically, in this case, theapplication device is removed when all the allergen will have diffused,or if the inflammatory reaction reaches a too strong intensity.

It is understood that, during the treatment, these alternatives may bealternated.

In an embodiment, a period of time comprised between 1 and 21 days isleft to elapse between the setting-up of devices.

The treatment may be considered as completed and successfully conductedwhen any cutaneous reactivity to the allergen will have disappeared orwill have been reduced very significantly. The success of the cure maybe confirmed by an oral elicitation test, or by any other meansrecognized in allergology. Accordingly, the duration of the cure isvariable and depends on the evolution of the cutaneous reactivity to theallergen. The objective of the desensitization cure according to themethod of the invention is either to cause the local cutaneous reactionto the allergen after prolonged contact to disappear entirely or toreduce the reactivity so that it is estimated that the reactivity of thesubject to the allergen has been significantly reduced by theepicutaneous immunotherapy cure, the risk of anaphylactic shock duringaccidental contact with the allergen is significantly reduced.

The invention may be applied with any cutaneous administration deviceensuring contact of the skin with the allergen. These are preferablydevices of the patch type, patches or bandages, preferably patches, forexample occlusive patches.

Such a device typically comprises a support on which the allergen isfixed, and/or a reservoir or a chamber comprising the allergen.

The device used is typically of transdermal nature with passivediffusion, i.e. it does not contain any means for causing perforation orabrasion of the skin. Indeed, the obtained data show that the method ofthe invention allows epicutaneous desensitization to be induced evenwithout preliminary skin treatment. However, it is also possible to usea device provided with properties aiming at increasing the permeabilityof the surface layers of the skin to the allergen and/or at increasingthe cutaneous reactivity with physical, chemical or biological means.Thus, in one alternative, the application device may act on thepermeability by means of micro-needles or electric means promoting thepassage of the allergen as far as the basal layer of the epidermis.Further, although this is not essential, it is possible to pre-treat theskin (for example by an abrasive treatment) before each application ofthe device in order to further promote the desensitization treatment.

In a preferred embodiment, a device of the occlusive patch type is used,such as for example an electrostatic patch Viaskin® described in patentapplication WO02/071950.

Moreover, in a preferred embodiment, the device is provided with meansensuring its maintaining on the skin. Any adhesive means may be used forthis purpose, including materials of the gum, polymer or plastic type,having strong adhesivity. It is also possible to use a device providedwith a bracelet, as described for example in application No. FR0753787,or with an exfoliation sheet, as described for example in applicationNo. FR0753265.

It is understood that the specific nature and form of the device may beadapted by one skilled in the art, the required property being to ensurecontact between the allergen and the skin at the application site.

In a particular embodiment, the treatment is performed by means of adevice provided with an at least partly transparent backing (for examplea VIASKIN® device), so that the inflammatory reaction (for exampleappearance of erythema) may be directly observed through the transparentmembrane. The backing of the device may even be adapted in order to helpthe patient in determining the cutaneous reaction threshold level fromwhich he/she will be able to remove the device. Thus, in a particularlyadvantageous embodiment, a device is used for which the backing has atransparent area on which is printed a monitoring pattern for example ared grid or red concentric circles. When the skin has not reacted, thedrawings are well visible and they are erased when the cutaneousreaction is obtained. Resorting to a transparent backing system has manyadvantages, and notably:

-   -   this avoids unnecessary prolonged contact with the allergen,        improves cutaneous tolerance, minimizes unpleasant secondary        effects of the prurit type and in fact improves acceptance and        observance;    -   this increases the performances of the method in terms of safety        and further minimizes the risks of systemic reaction which have        been fatal to many methods for desensitization to groundnuts in        the past;    -   this provides a solution with which the notion of variable        exposure time to the allergen may be integrated into the        industrial process.

In this context, an object of the invention relates to a cutaneousdevice for applying an allergen on the skin of a subject, characterizedin that it comprises a backing provided with a transparent area throughwhich the inflammatory cutaneous reaction elicited by the application ofthe allergen on the skin may be viewed. Advantageously, the device is adevice of the occlusive type and/or with passive diffusion. The deviceis typically a patch.

Another object of the invention moreover relates to a kit comprisingseveral devices for cutaneous application of an allergen, the devicescomprising different doses of the allergen and/or different forms of theallergen.

The allergen used within the scope of the invention may be of variousnature, form and origin. Thus, it may be a food, respiratory orcutaneous allergen, for example. These may be proteins (or polypeptidesor peptides), lipids, sugars, etc., which may be in the form ofextraction products, in a recombinant form, and/or of synthetic origin,for example if necessary, chemically or immunologically modified inorder to modify their properties. The allergens may be in an entire,native form, or in a fragmented, denaturated form, etc. A preparation istypically used, comprising several allergenic antigens as a combination,and/or allergen(s) as conjugates or complexes. It is understood that themethod of the invention is essentially independent of the nature of theallergen, and may therefore be applied to any preparation comprisingallergens, such as for example food allergens (e.g. groundnut,shellfish, milk, egg, fish, wheat, soya bean, etc.) or cutaneous orrespiratory allergens (mites, pollen, animal hairs, etc.).

The allergen is advantageously formulated with any excipient or carrieradapted to pharmaceutical use. The applied allergenic preparation mayappear in different forms, such as for example as a liquid or solid. Ina preferred embodiment, the allergen is in dry form, i.e. in dehydratedform, and notably as a powder, particles, agglomerates, a wafer, driedform, etc. The dry form may be obtained by freeze-drying, of by simpleevaporation.

In a particular embodiment, the allergen is in the form of a powder.

As indicated earlier, an advantage of the invention it that with it, itis possible to obtain desensitization to an allergen even withoutco-administration of an adjuvant. Also, in a preferred embodiment, theallergenic preparation applied on the skin of a patient, is free of anyadded adjuvant compound. The term adjuvant in the sense of the inventiondesignates any compound which exerts physico-chemical and/or biologicalaction on the skin, facilitating penetration of the antigen into theskin, and which increases the immune response against the antigen. Byphysico-chemical and/or biological action, the adjuvant, as definedhere, is a compound which alters the permeability of the epidermis bydisturbing the corneal layer, thereby allowing passage of the antigenand its contact with the immunologically active cells of the epidermis.Examples of adjuvant compounds are notably choleric toxin (CT) orthermolabile toxin (LT) of E-coli. Other examples are transfersomes,described by Paul et al. (Paul A, Cevc G, Bachhawat BK. Transdermalimmunization with large proteins by ultradeformable drug carriers. Eur.J. Immunol. 1995, 25 (12): 3521-4), which allow transport of an antigenin the membranes.

Nevertheless, in one alternative, the allergen may be used as acombination with an adjuvant compound and/or provided with propertiesfor amplifying the inflammatory reaction to said allergen, for examplewith adjuvants or compounds with immuno-modulating activity, such as forexample a cytokine, a compound, Toll receptor ligands, etc. Theexpression <<used as a combination>> indicates that the allergen and thecompound are administered to the same subject in order to induce acooperation effect in vivo. Nevertheless, they may be administeredaccording to various procedures and routes. Thus, they may be bothapplied via an epicutaneous route, or else the compound is applied viaanother route. Moreover, they are not necessarily applied at the sametime, but may be applied sequentially, repeatedly or not, and at similarfrequencies or not. Typically, the adjuvant compound is administeredbefore or at the same time as the allergen.

In a normal embodiment, the device contains an amount of allergenscomprised between 0.1 and 1,000 μg/cm² of the surface area of thedevice, preferably between 20 and 500 μg per cm², more preferentiallybetween 20 and 200 μg per cm². It is understood that this amount may beadapted by one skilled in the art depending on the goals, the pathology,the duration of the treatment, the subject, etc.

The experimental examples which follow show that such a reduction inreactivity may be attained by using a control of the doses according tothe invention. The epicutaneous immunotherapy method of the inventionincludes many advantages relatively to the present state of the art.

This is an original method aiming at eliciting and controlling aninflammatory reaction by regularly and durably applying an allergen onthe skin of a subject allergic to said allergen with the purpose ofdirecting the reaction of the body against this allergen towardstolerance.

This is a perfectly safe method as confirmed by cumulated experience inthe field of atopy patch tests as well as by data collected since theintroduction of the DIALLERTEST® in France, a first ready-to-use atopypatch test with more than 100,000 distributed copies. Because of thisgreat safety of use, with the method according to the invention, it ispossible to desensitize subjects allergic to allergens such asgroundnuts or shellfish, which are not managed by standarddesensitization methods because of the incurred risks.

Immunotherapy may be interrupted at any moment by removing the deviceapplied on the skin, which cannot be contemplated with traditionaldesensitization methods during which the antigen is administered intothe body.

With the first step of the method, it is possible to confirm eligibilityof the subject for epicutaneous immunotherapy (the subjects having acutaneous reaction are eligible) on the one hand, and to measure thedegree of cutaneous reactivity of the subject to the allergen on theother hand, in order to at best guide the management of the epicutaneousimmunotherapy cure. It is thus possible to select the procedure which isthe most adapted to the patient in order to optimize cutaneous toleranceand to maintain the best level of effectiveness.

During the cure, it is possible to adjust the dosage and/or thefrequency of administration depending on the evolution of the cutaneousreactivity, as measured continuously. It is thus possible to permanentlyoptimize the therapeutic action and to maintain the highest level ofeffectiveness all along the treatment.

The duration of the cure depends on the therapeutic objectives set atthe beginning of the cure and on the evolution of the cutaneousreactivity to the allergen. The cure is considered as completed when theobjectives of reducing reactivity to the allergen as ascertained on theskin are achieved and confirmed by an elicitation test or any otherallergology means or when complete extinction of cutaneous reactivity isascertained.

Other aspects and advantages of the invention will become apparent uponreading the examples which follow, which should be considered asillustrative and non-limiting.

CAPTIONS OF THE FIGURES

FIG. 1: plethysmography results obtained after 8 weeks (a) or 16 weeks(b) for desensitization to the pollen of Dactyle. C: control, ND:non-desensitized, SCD: subcutaneous desensitization, EPD: epicutaneousdesensitization.

FIG. 2: plethysmography results obtained after 8 weeks (a) or 16 weeks(b) for desensitization to ovalbumin. C: control, ND: non-desensitized,SCD: subcutaneous desensitization, EPD: epicutaneous desensitization.

FIG. 3: monitoring of the antibody level before and after 8 weeks ofdesensitization with groundnut proteins. Left graph: IgE specific, rightgraph: IgG1 and IgG2a specific. EP: epicutaneous, ID: intradermal, ND:non-desensitized.

FIG. 4: histamine released after an elicitation test. EP: epicutaneous,ID: intradermal, ND: non-desensitized. The results are expressed in nM.

FIG. 5: evolution of the rate of specific IgEs (V0, initial sample, V1,samples at one month, V3, sample at 3 months).

FIG. 6: viewing the cutaneous inflammatory reaction during thetreatment: A: marked; B: medium; C: attenuated.

FIG. 7A: activation of Langerhans cells and inflow of inflammatory cellsafter 24 hrs and 48 hrs of application of an allergen with VIASKIN®.After 24 hrs of application, the Langerhans cells are activated andbegin their migration towards the lymph nodes. After 48 hrs, theinflammatory cells are alerted and inflammatory granulomas are wellvisible.

FIG. 7B: evolution of the local cell response after differentapplication times of an allergen (ovalbumin) with VIASKIN®. After 6 hrsof application, the cell response is not perceptible. After 24 hrs, therise in the local rate of inflammatory cells continues after removingVIASKIN®. This evolution is stronger after 48 hrs of application.

FIG. 8: evolution of the local response profile after prolongedapplication (6 and 12 weeks) of an allergen on the skin of an animalsensitized with VIASKIN®. After repeated application for 24 hrs, theresponse is predominantly of type TH2. After repeated applications for48 hrs, the response is mixed, and then predominantly TH1.

EXAMPLES Example 1. Desensitization to Ovalbumin and to Pollen

1.1. Methodology

In the case of OVA and POL, BALB/c mice were sensitized for 2 weeks insuccession (D0 and D7) by subcutaneous injection of 10 μg of allergenassociated with aluminium hydroxide. At D14, the mice received nasalinstillation of 10 μg of allergen alone.

From D21, the mice either followed a desensitization treatment or not.The treated mice received once a week for 8 weeks, 100 μg of allergenadministered either via an epicutaneous route by means of the VIASKIN®system, under conditions ensuring sustainment of the cutaneousinflammatory reaction, or via a subcutaneous route in association withaluminium hydroxide (positive control). The non-desensitized mice weremaintained under normal conditions of an animal house.

The evolution of the antibody (IgE and IgG specific) levels was trackedon sera from ice, sampled after 8 weeks and 16 weeks of desensitization.

The effectiveness of OVA and POL desensitization treatment was alsostudied by plethysmography measurements allowing evaluation of bronchialhyperreactivity of mice either desensitized or not.

1.2. Results

Bronchial hyperreactivity was measured for mice either treated or notwith VIASKIN® (FIGS. 1 and 2).

Bronchial hyperreactivity of the mice treated with VIASKIN® underconditions ensuring sustainment of the cutaneous inflammatory reaction,is significantly reduced, by 3.7 to 2.6 times relatively to untreatedmice. Bronchial hyper reactivity of the mice treated subcutaneously isnot significantly different from that of mice treated with VIASKIN®.

Example 2. Desensitization to Groundnuts

2.1. Methodology

For ARA (groundnuts), the mice were sensitized over 6 weeks byintragastric administration every 6 days (D1, D7, D13, D19, D25, D33) of1 mg of ARA proteins associated with 10 μg of choleric toxin.

The desensitization procedure begins at D75 for a period of 8 weeks. Themice are desensitized once a week by administering 100 μg of ARAformulation via an intradermal route or via an epicutaneous route withthe system VIASKIN®, under conditions ensuring sustainment of theinflammatory reaction. Non-desensitized mice do not follow anyparticular treatment after sensitization.

The effectiveness of desensitization is tracked by measuring theantibody (IgE, IgG1 and IgG2a specific) level as well as by measuringthe release of histamine after oral elicitation tests. Histamine is amediator released during degranulation of immune cells of the mastocytesor basophilic type. This degranulation is a marker of the induction ofthe allergic response.

2.2. Results

The analysis of the ARA desensitization treatment was validated byanalysis of the specific antibody levels in mice (FIG. 3). The micedesensitized via an epicutaneous route or via an intradermal route havea reduction in the level of specific IgE (marker of allergy to ARA) andan increase in the specific IgG2a level. Specific IgE levels detectablein non-desensitized mice remain high and specific IgG2a levels are lowerthan in desensitized mice. No significant difference was observedbetween the epicutaneous and intradermal desensitization routes.

Oral elicitation tests were conducted on mice either desensitized ornot. The mice desensitized via an intradermal route or via anepicutaneous route have lower release of histamine than non-desensitizedmice. This result shows a decrease in the allergic reactivity ofdesensitized mice.

Discussion

Through the different study models and the different studied allergens,it appears that:

-   -   the epicutaneous immunotherapy method based on sustainment of        the inflammatory response enables triggering of a clinical and        biological response of the systemic type;    -   the animals having benefited from the epicutaneous immunotherapy        method based on sustainment of the inflammatory response after        preliminary sensitization via an oral or cutaneous route have an        attenuated response to elicitation tests, comparable with that        of animals having been subject to immunotherapy via a        subcutaneous or intradermal route;    -   the obtained serological response confirms induction of a        tolerance process by epicutaneous immunotherapy.

Example 3. Clinical Data

Characteristic of the Patient:

child, 6 years 5 months old, 18.3 kg. Chronic diarrhea, vomiting,generalized eczema. Positive prick test to cow milk. Total IgE 486 IU.Cow milk specific IgE 32.7 IU.

Positive milk elicitation test at 0.6 mL (laryngeal prurit, coughing,abdominal pains, urticaria).

Treatment:

Application of VIASKIN® containing 130 μg of milk proteins, renewedevery two days for 3 months under conditions ensuring persistence of acutaneous reaction upon contact with the allergen.

Results:

The results are shown in FIGS. 5 and 6. They illustrate very substantialimprovement in the tolerance threshold to cow milk, correlated withmonitoring of the inflammatory reaction during the treatment. At the endof the immunotherapy cure, attenuation of the cutaneous reaction isascertained. The oral elicitation test at 3 months shows a tolerancethreshold up to 64.6 mL without any clinical reaction.

Example 4. Evolution of the Cell Response Following Application of anAllergen in Sensitized Mice

Application of an allergen on the skin of the mouse sensitizedbeforehand activates Langerhans cells and their migration towards lymphnodes (FIG. 7A).

Within the same time, the activated cells produce pro-inflammatorycytokines which attract the cells of the inflammation towards thesurface layer of the skin. This <<first phase>> reaction occurs insensitized subjects since it has memory cells and specific IgEs directedagainst the allergen. Therefore, upon contact with the allergen, thereoccurs an initial reaction of rejection of the allergen.

In a second phase, a local inflammatory reaction characterized by theinflow of acute inflammation cells (mastocytes, macrophages) and chroniccells (leukocytes), appears (FIGS. 7A and 7B). In mice sensitizedbeforehand, application of an allergen (ovalbumin) on the skin withViaskin® is expressed by a slightly inflammatory reaction after 24 hrsof contact and on the other hand by a massively inflammatory reactionafter 24 hrs of contact with invasion of the cutaneous area bypolynuclears. It is this more complex reaction which is targeted by thetreatment.

In an investigation conducted in sensitized mice, the application of theallergen 6 hrs, 24 hrs and 48 hrs determines an inflammatory reaction(materialized here by local increase in the level of macrophages) whichis more significant after 48 hrs of application (FIG. 7B).

Discussion

During the epicutaneous desensitization treatment, the aim is toreproduce an inflammatory cell reaction, the role of which isindispensable in the tolerance process.

In reality, it is not the intensity of the inflammatory reaction whichvaries over time but especially its very nature.

Immunofluorescence studies confirm the central role of the inflammationand the completely different aspect of the profile of the inflammatoryreaction depending on the application time.

The application time of the allergen is an essential factor foreliciting a cell reaction.

Example 5. Evolution of the Local Response Profile after ProlongedApplication of an Allergen

The application time is not a sufficient condition for obtaining thetype of sought therapeutic (tolerogenic) reaction. In mice, repeatedadministration of an allergen on the skin causes a rise in the specificantibodies of this allergen regardless of whether the application is<<short>> (24 hrs) or <<long>> (48 hs). When the application iscontinued (up to 88 days), the level of the antibodies is still alsohigh but the relative proportion of Th1/Th2 lymphocytes is modified inthe direction of a rise in Th1 lymphocytes in subjects receivingViaskin® for 48 hrs (FIG. 8).

Discussion

Experimental data demonstrate that the method according to the inventionis aimed at inducing a reaction, the characteristics of which aredifferent from the reaction usually obtained by simply depositing theallergen on the skin of allergic persons. It is only the reactionobtained after <<long>> application by means of Viaskin®, with which a<<tolerogenic>> effect may be obtained, materialized by an activation ofTh1 lymphocyte cells while the reaction obtained after a <<short>>application is of the Th2 type.

During the desensitization cure by Viaskin®, repeated application of theallergen on the intact skin of the patient sensitized beforehand,elicits an inflammatory cell reaction as currently observed clinicallywith atopy patch tests.

By eliciting and sustaining the inflammatory reaction by repeated andprolonged application of the allergen, specific local inflammationconditions are created. During this reaction, the equilibrium is shiftedin favor of the Th1 lymphocytes.

It therefore appears that prolonged and repeated application of anallergen on the skin is not only capable of causing an inflammatoryreaction, but also of orienting this reaction.

1. A method for epicutaneous desensitization of a subject allergic tomilk, the method comprising: a) determining a dose of a milk allergenwhich causes a local inflammatory reaction on the skin of the subjectupon epicutaneous application thereof; b) epicutaneously exposing thesubject to the milk allergen and generating the local inflammatoryreaction in the subject by applying said dose of the milk allergen,without an adjuvant, onto an intact region of skin of the subject; andc) repeatedly applying the milk allergen, without an adjuvant, to anintact region of the skin of the subject, at a dose, frequency or for anapplication time adapted to sustain a local inflammatory reaction, theexposure and sustained reaction leading to desensitization of thesubject; wherein the milk allergen is applied by a cutaneous occlusivedevice configured to ensure contact between the allergen and the regionof skin, and wherein said dose of milk allergen is comprised between 0.1and 1,000 μg/cm² of a surface area of the device.
 2. The method of claim1, further comprising visually evaluating the inflammatory reaction. 3.The method of claim 1, wherein the dose, frequency, or application timeof the milk allergen is increased as the inflammatory reaction decreasesduring the repeated applications.
 4. The method of claim 1, wherein thedose, frequency, or application time of the milk allergen is decreasedas the inflammatory reaction increases during the repeated applications.5. The method of claim 1, wherein the milk allergen is in powder form.6. The method of claim 1, wherein the inflammatory reaction generatedduring the generating step is visible to a naked eye.
 7. The method ofclaim 6, wherein the inflammatory reaction comprises an infiltratederythematous lesion.
 8. The method of claim 6, wherein the inflammatoryreaction has an eczematiform aspect.
 9. The method of claim 1, whereinthe device comprises a transparent backing configured to allow theinflammatory reaction to be viewed.
 10. The method of claim 9, whereinthe backing comprises a grid configured to aid in an evaluation of theinflammatory reaction.
 11. The method of claim 1, wherein the device isremovable.
 12. The method of claim 1, wherein step a) comprisesdetermining the inflammatory reaction of the subject generated bydifferent doses of the milk allergen.
 13. The method of claim 1, whereinthe milk allergen comprises milk proteins.
 14. The method of claim 1,wherein step a) further comprises: determining the inflammatory reactionof the subject generated by different forms of the milk allergen; andselecting a form of milk allergen for the treatment based on saiddetermination.